Plasmid Midi Kit II, Biomiga

Biomiga's Plasmid Purification System

·         Superior Purity:This patented Plasmid Purification system uses no chaptropic salts in the buffer, and the resulting purified DNA is free of guanidine salts, cesium chloride, ethidium bromide and ion exchange resin residues, which enable the highest performance of downstream applications including gene therapy and genetic vaccination.

·         Ultra High Yield:When comparing Biomiga' Plasmid Purification System to other purification methods, our methodology produces higher DNA yield while requiring fewer bacterial cultures.

·         Time Saving:Using our unique Megaprep Kit, 10 mg plasmid DNA can be purified from 1,000 mL culture within 1 hour!

·         Environment benefit:The EZgene System is the only method available that does not utilize Guanidine Salt, a hazardous protein denaturant, in the silica based purification system, which will ensure the safety and health of scientists and the environment!

 

Citations

Murine Xenograft Model for Human Uterine Fibroids: An In Vivo Imaging Approach.  Reproductive Sciences, 2009, 16(9), 827-842

Characterization of monoclonal antibodies against goat interleukin-18 and their application in the measurement of goat interleukin-18 in LPS-stimulated peripheral blood mononuclear cells by sandwich ELISA. Veterinary Immunology and Immunopathology, Volume 138, Issue 3, 2010, 235-238

Platelet-Derived Growth Factor C Is Upregulated in Human Uterine Fibroids and Regulates Uterine Smooth Muscle Cell Growth1. Biology of Reproduction, 2009, vol. 81 no. 4749-758

Cloning, characterization and tissue distribution of a pi-class glutathione S-transferase from clam (Venerupis philippinarum): Response to benzo[?]pyrene exposure. Comparative Biochemistry and Physiology, Part C: Toxicology & Pharmacology , 2010,Volume 152, Issue 2, August 2010, Pages 160-166

An Mpeg (macrophage expressed gene) from the Pacific oyster Crassostrea gigas: Molecular characterization and gene expression.Fish & Shellfish Immunology, 2011, Volume 30, Issue 3, March 2011, 870-876

Construction of eukaryotic recombinant vector of renalase and its expression as a eukaryotic protein.Journal of Shanghai Jiaotong University (Science), 2010, Volume 15, Number 5, 637-640

 

Biomiga Plasmid Midi Kit

Biomiga Plasmid Midi Kit provides an easy and reliable method to purify plasmid DNA from 50-100 mL bacterial cultures in less than 60 minutes. The DNA is ready for downstream applications such as transfection, RFLP, DNA amplification, and automated sequencing.

  • Biomiga Plasmid Midi Kit I: 100-200 μg of high copy number plasmid DNA can be isolated from 50 mL culture.
  • Biomiga Plasmid Midi Kit II: 200-400 μg of high copy number plasmid DNA can be isolated from 100 mL culture.
  • Choice of 2 types of columns for centrifugation or vacuum manifold.

·        Plasmid ezFilter Midi Kit , Centrifuge

·        Plasmid  ezFilter Midi Kit, Vacuum Manifold

  • Endofree Plasmid Midiprep Kit: Endotoxin < 0.2 EU / μg DNA.

 

Biomiga Plasmid Midi Kit II

The key to Biomiga Plasmid Midi Kit II is its proprietary DNA binding systems that allow the highly efficient binding of DNA to our ezBindTM matrix while proteins and other impurities are removed by Wash Buffer. Nucleic acids are easily eluted with sterile water or Elution Buffer.

Unlike other kits in the markets, no chaotropic salts are contained in the buffer of our patented plasmid purification kit. The purified DNA is guanidine/anion exchange resin residues free.

This kit is designed for fast and efficient purification of plasmid DNA from 50 to 100 mL of E. coli culture. The midi column has a plasmid DNA binding capacity of 500 µg.

The purified DNA is ready for high performance of downstream applications such as transfection of robust cells such as HEK293, restriction mapping, library screening, sequencing, as well as gene therapy and genetic vaccinations.

 

Important Notes

Plasmid Copy Numbers: The yield of plasmid DNA depends on the origin of the replication and the size of the plasmid. The protocols are optimized for high copy number plasmid purification. For low copy number plasmids, both the culture volume and the buffer volume need to be scaled up 2 times. Please contact our customer service for further information and reference Table 1 for the commonly used plasmids,

Table 1 Commonly used plasmid and expected yield.

Plasmid

Origin

Copy Numbers

Expected Yield(µg per  50 mL)

pSC101

pSC101

5

5

pACYC

P15A

10-12

5-10

pSuperCos

pMB1

10-20

10-20

pBR322

pMB1

15-20

10-20

pGEMR

Muted pMB1

300-400

100-150

pBluescriptR

ColE1

300-500

100-200

pUC

Muted pMB1

500-700

150-250

Host Strains: The strains used for propagating plasmid have significant influence on yield. Host strains such as Top 10 and DH5a yield high-quality plasmid DNA. endA+ strains such as JM101, JM110, HB101, TG1 and their derivatives, normally have low plasmid yield due to either endogenous endonucleases or high carbohydrates released during lysis. We recommend transform plasmid to an endA- strain if the yield is not satisfactory. For purifying plasmid DNA from endA+ strains (Table 2), we recommend use product PD1712.  

 

Table2 endA strains of E. Coli.

EndA- Strains of E. Coli

DH5α

DH1

DH21

JM106

JM109

SK2267

SRB

XLO

TOP10

DH10B

JM103

JM107

SK1590

MM294

Stbl2TM

XL1-Blue

BJ5182

DH20

JM105

JM108

SK1592

Select96TM

Stbl4TM

XL10-Gold

EndA+ Strains of E. Coli

C600

JM110

RR1

ABLE® C

CJ236

KW251

P2392

BL21(DE3)

HB101

TG1

TB1

ABLE® K

DH12STM

LE392

PR700

BL21(DE3)pLysS

JM101

JM83

TKB1

HMS174

ES1301

M1061

Q358

BMH 71-18

All NM  strains

All Y strains

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Optimal Cell Mass (OD600 x mL of Culture): This procedure is designed for isolating plasmid grown in standard LB medium (Luria Bertani) for 12 -16 hours to a density of OD600 2.0 to 3.0. If rich mediums such as TB or 2xYT are used, make sure the cell density doesn't exceed 3.0 (OD600). A high ratio of biomass over lysis buffers result in low DNA yield and purity. The midi II column has an optimal biomass of 150-250. For example, if the OD600 is 3.0, the optimal culture volume should be 50 to 80 mL.

 

Culture Volume: Use a flask or tube 4 times bigger in volume than the culture medium to secure optimal condition for bacteria growth. Don't exceed the maximum culture volume suggested in the protocol. Incomplete lysis due to over amount of bacterial culture results in lower yield and less purity.

 

Storage and Stability

Buffer A1 should be stored at 4°C once RNase A is added. All other materials can be stored at room temperature (22-25°C). The Guaranteed shelf life is 12 months from the date of purchase.

 

Before Starting

Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each step.

 Important

  • RNase A: It is stable for more than half a year when stored at room temperature. Spin down the RNase A vial briefly. Add the RNase A solution to Buffer A1 and mix well before use. Store at 4°C.
  • Buffer B1 precipitates below room temperature. It is critical to warm up the buffer at 50°C to dissolve the precipitates before use.
  • Keep the cap tightly closed for Buffer B1 after use.
  • Make sure the availability of centrifuge (13,000 rpm). Especially, after mixing the lysate with ethanol, the sample needs to be processed immediately by centrifugation.
  • Carry out all centrifugations at room temperature.

 

Materials supplied by users

  • 70% ethanol and 100% ethanol
  • High speed centrifuge
  • 30 mL high speed centrifuge tubes
  • 50 mL conical tubes
  • Isopropanol if precipitate the plasmid DNA.

 

Kit Contents

Catalog #

PD1412-00

PD1412-01

PD1412-02

Preps

2

10

25

ezBindTM Columns

2

10

25

Buffer A1

11 mL

55 mL

135 mL

Buffer B1

11 mL

55 mL

135 mL

Buffer C1

14 mL

70 mL

170 mL

Buffer KB

9 mL

50 mL

110 mL

RNase A (20 mg/mL)

1.1 mg(55 µL)

5.5 mg(275 µL)

13.5 mg(675 µL)

Elution Buffer

 4 mL

20 mL

60 mL

User Manual

1

1

1

 

Safety Information

Buffer C1 contains acetic acid, wear gloves and protective eyewear when handling.

 

Operating Protocol